RNA polymerases are enzyme molecules well-known to those skilled in the fields of molecular biology and molecular diagnostic kits. RNA polymerases synthesise RNA molecules from a DNA template strand.
Much research has been carried out on RNA polymerases, especially bacteriophage RNA polymerases.
Specifically, the RNA polymerase from the bacteriophage T7 has been shown to be very selective for specific promoters that are rarely encountered in DNA unrelated to T7 DNA (Chamberlin et al, 1970 Nature 228, 227; Dunn & Studier 1983 J. Mol. Biol. 166, 477). T7 RNA polymerase is able to make complete transcripts of almost any DNA that is placed under control of a T7 promoter. T7 RNA polymerase is a highly active enzyme that transcribes about five times faster than does Escherichia coli RNA polymerase (Studier et al, 1990 Methods Enzymol. 185, 60). The synthesis of small RNAs using T7 RNA polymerase has been described whereby sequences around the RNA polymerase promoter sequence are shown to be important in the reproducible improvement of yield of RNA produced (Milligan & Uhlenbeck, 1989 Methods Enzymol. 180, 51 and Milligan et al, 1987 Nucl. Acids Res. 15, 8783–8798). Other RNA polymerases that have similar properties to T7 include those from bacteriophage T3 and SP6, the genes for which have all been cloned and the corresponding enzymes are commercially available. The optimum promoter sequences for T7, T3 and SP6 polymerases are known, and are essentially 17 nucleotides long.
A number of methods have been disclosed, which utilise RNA polymerases to synthesise RNA directly or indirectly as the result of the presence of a particular nucleic acid sequence of interest (“target”). The presence of RNA (detected directly or indirectly) thus serves to signal the presence of the sequence of interest and can be used as the basis of assay methods and/or diagnostic methods or kits. Examples include the disclosures of WO 93/06240, WO 94/29481, EP 0851033, and EP 0552931.
In particular WO 93/06240 discloses the use of two probes, which hybridise together only in the presence of a target nucleic acid sequence of interest, such that hybridisation of the probes to each other is indicative of the presence of the sequence of interest.
All publications mentioned in this specification are incorporated herein by reference.